Diagnosis or post-treatment diagnosis of leukemia, pre-leukemia or aleukemic malignant blood diseases is important for determining the strategy for treating these diseases.
For diagnosing leukemia at its first occurrence, there is a method in which the white blood cell count in the peripheral blood of a patient is determined. When the white blood cell count is beyond the normal level, the occurrence of leukemia is suspected. However, the white blood cell counts also increase due to the enhancement of the immune-response within the body in the case of diseases other than leukemia, such as a cold. Hence, determination of the white blood cell counts alone may allow false-positive results. Besides, normal white blood cell counts in the peripheral blood range as broad as 4,000-8,000 cells/μL, so that false-negative cases are possibly raised. Therefore, a method for diagnosing leukemia with higher accuracy is in need.
As a method for diagnosing recurrence of leukemia, detection by RT-PCR of the WT-1 gene is mentioned [Clinical Pathology, 48, (underlining in original) 155 (2000), Blood, 84, (underlining in original) 3071 (1994), Japanese Patent No. 3122771]. This diagnostic method is complicated in handling, and requires a special device. As therapies for leukemia, pre-leukemia or aleukemic malignant blood diseases mentioned above, congenital metabolic diseases, solid cancers and the like, the hematopoietic stem cell transplantation therapy is mentioned. Drawbacks associated with the hematopoietic stem cell transplantation therapy include the HLA-type incompatibility between the hematopoietic stem cells of a donor and a patient, insufficient effect of the therapy owing to the physical condition of a patient and to infection, etc., such as non-engraftment of the transplanted hematopoietic stem cells, delayed engraftment of the hematopoietic stem cells, occurrence of graft versus host disease (hereinafter referred to as GVHD). When things turn to the worst, it may proceed to death.
Delayed engraftment of the hematopoietic stem cells can be coped with by the in-vivo administration of G-CSF to promote the engraftment of the hematopoietic stem cells. To cope with GVHD, in-vivo administration of immunosuppressants also suppresses rejection of the transplanted hematopoietic stem cells. However, there is a fear of side effects for either kind of agents, if administered in an excess amount. Therefore, it is crucial for determining the treatment strategy to diagnose or predict the engraftment of the hematopoietic stem cells or the occurrence of GVHD.
As a method of confirming the engraftment of hematopoietic stem cells after transplantation of the hematopoietic stem cells, there is a method in which white blood cell counts or platelet counts in the peripheral blood are determined. Hematopoietic stem cells can be diagnosed as being engrafted if these levels are increased. However, it sometimes requires from 10 days to a month or longer after transplantation for the hematopoietic stem cells to engraft, so that the engraftment of hematopoietic stem cells cannot be diagnosed at an early stage by only determining white blood cell counts or platelet counts in the peripheral blood.
A method for diagnosing the occurrence of GVHD includes observing skin rush or the like emerging at a recovery phase after transplantation of the hematopoietic stem cells. However, an easy and accurate method for diagnosing the occurrence of GVHD remains unknown. Moreover, any method to predict the occurrence of GVHD prior to its occurrence is unknown.
Human stem cell growth factor (hereinafter abbreviated as SCGF) is a protein comprising an amino acid sequence of SEQ. ID No. 1 or 2 [WO98/08869, Proc. Natl. Acad. Sci. USA, 94, 7577 (1997), Biochem. Biophys. Res. Comm., 249, (underlining in original) 124 (1998)].
Among antibodies known to recognize SCGF, there are polyclonal antibodies which are prepared by using SCGF obtained by genetic recombination and a partial peptide of SCGF which consists of 6-25 amino acid residues from the N-terminus, as immunogens, and monoclonal antibodies which are prepared by using SCGF purified partially from the cell culture supernatant and SCGF obtained by genetic recombination, as immunogens [WO98/08869]. The reference reports that this monoclonal antibody has a neutralizing activity, that a polyclonal antibody, which is prepared by using SCGF obtained by genetic recombination as an immunogen, reacts with SCGF obtained by genetic recombination when subjected to ELISA, and that SCGF obtained by genetic recombination can be detected by western blotting using a polyclonal antibody prepared by using a partial peptide of SCGF consisting of 6-25 amino acid residues from the N-terminus as an immunogen.
Further, there is a report as to anti-SCGF monoclonal KM2142 antibody which is prepared by using a partial peptide of SCGF consisting of 6-25 amino acid residues from the N-terminus as an immunogen [The Hematology Journal, 2, (underlining in original) 307 (2001)].
It is known that the expression levels of the SCGF gene, as revealed by northern blotting for human normal tissues, are high in the kidney, low in the heart, and nil in the brain, placenta, lung, liver, skeletal muscles and pancreas [Proc. Natl. Acad. Sci. USA, 94, (underlining in original) 7577 (1997)], high in the spleen, thymus, cecum, bone marrow, fetal liver and low in peripheral blood [Biochem. Biophys. Res. Comm., 249, (underlining in original) 124 (1998)]. Also, it is reported that, as a result of in-situ hybridization with normal new-born mice, SCGF is expressed in the bone marrow, proliferating cartilage, and in the proximal periosteum [The Hematology Journal, 2, (underlining in original) 307 (2001)].
It is further reported that while the SCGF gene expression is observed in bone marrow cell lines (HT60, KPB-M15), monocyte cell lines (THP-1, U-937), an erythroblast cell line (HEL) and a fibroblast cell line (NHF), expression of the gene is not observed in B-cell lines (U266B1, IM-9), a T-cell line (MOLT-4), an erythroblast cell line (K562), epithelial cancer cell lines (HeLaS3, A431), a melanoma cell line (Bowes), an adenovirally transformed fetal kidney cell line (293) and a fibroblast cell line (CCD-8Lu) [Proc. Natl. Acad. Sci. USA, 94, 7577 (1997)].
There has been, however, no reports with regard to differences in mRNA amounts of SCGF in the peripheral blood and in the bone-marrow blood cells from animals including humans that are either normal or suffering blood diseases, or from those that underwent transplantation of the hametopoietic stem cells.
Since the mRNA levels of tissues and cells have low correlation with the encoded proteins (correlation coefficient=0.48) [Electrophoresis, 18, 533 (1997)], it is a hard task to estimate the level of SCGF protein from that of SCGF mRNA.
Thus, the existence, function and association with diseases of SCGF protein in the body fluid such as serum and plasma and in the tissues from animals including humans are left unrevealed.
An object of the present invention is to provide a method for diagnosing leukemia, pre-leukemia or aleukemic malignant blood diseases, a method for discriminating leukemia from pre-leukemia or aleukemic malignant blood diseases, a method for discriminating aplastic anemia from myelodysplastic syndrome, and a method for diagnosing the engraftment of hematopoietic stem cells and GVHD after transplantation of the hematopoietic stem cells, and to provide an agent and a kit for diagnosing leukemia, pre-leukemia or aleukemic malignant blood diseases, and an agent and a kit for diagnosing the engraftment of hematopoietic stem cells and GVHD after transplantation of the hematopoietic stem cells.